Effects And Mechanisms Of Ketamine On Apoptosis Of The Developing Hippocampal Neurons In Rats

Partâ…  Serum-free cultivation, identification and morphologicalobservation of primary hippocampal neurons from newborn ratsObjective: To establish a simple and practical method of the serum-freeprimary culture of hippocampal neurons in vitro to obtain highly purified andenergetic neurons;To observe the morphological characteristics and detect thegrowth curve of hippocampal neurons.Methods: New-born SD rats in24h were sacrificed by decapitation, andthe brains were carefully removed and transferred into the fluid of precooledD-hanks. Hippocampi were dissected from the brains. The small tissue pieceswere dissociated mechanically and digested with ACCUTASE for15-20min.The reaction of ACCUTASE was stopped with DMEM involved in10%FBS,and cells were recovered after5min of centrifugation at800r/min. Single cellsuspension was obtained by svsieving cells with a200mesh cell sieve.Cellswere transferred into plastic culture flasks and the latter were put into theincubator for1h in order to harvest the neurons whose adherence speed wasslower than glial cells'. Before planted onto the Metrigial-coated in glasscovers and then put into the incubator, the neurons were counted by trypanblue staining and the densitiy of neurons were diluted into1×106/mL. Themediums containing serum were replaced by the serum-free Neurobasalmedium supplemented with B27and N2within24h. Half of the culturemedium was changed every2days thereafter. The morphologicalcharacteristics of primary hippocampal neurons were observed under thephase-contrast microscope.On the seventh day, the purity of the neurons wereidentified by the immunofluorescence chemistry of β-tublinШ. The growthcurve of hippocampal neurons were drawn by measuring MTT absorbanceevery2days. Results:1. The newly inoculated hippocampal neurons were round,small, translucent, and in a single suspension uniform distribution. After3h,the cells started to adhere to the culture plates, and most of neurons hadadhered in20h. Their morphologies were fusiform, triangular, with differentlength of slender protrusions. On the3th day, the cells were well-rounded andfull, and the prominences were elongated and enlarged more significantly andconnected to the network. Sparse connections had come into being. From the7th d to10th d, the cell body became larger than before and appeared obvioushalo. Furthermore, network became denser. The cell body was multipolar.There were many kinds of shapes, such as pyramid, triangle, spindle andellipse. After cultivated for20d, the cells began to degenerate.The halos of thecells body were disappeared. The phenomena of the prominence regressionand nuclus pycnosis could be observed.2. The primary cultivatedhippocampal neurons were verified by positive staining of β-tublinШ againstHoechst33258. The purity of the hippocampal neurons is94.2%±3.6%.3. Theprimary cultivated hippocampal neurons undergone the latency phase (2-4d),the exponential growth phase (4-14d), and then the growth plateau phase(14-16d), in which the growth of cell became retard nearly.Conclusions:1. The primary cultivated neurons are mature on the10th d.The cell bodies are triangle, spindle and oval under the phase inverted contrastmicroscope.2. The immnunocytochemistry of cultivated hippocampal neuronsis showed with the positive of β-tublinШ against Hoechst33258. The positivecell ratio was94.2%±3.6%.3. The cultivated neurons undergo a latency phase,exponential growth phase and plateau phase in vitro.Partâ…¡ Effects of ketamine on the viability and apoptosis of thedevelopping hippocampal neuronsObjective: To evaluate the effects of ketamine with differentconcentrations and cultivating time on the viability of developping primaryhippocampal neurons in vitro by MTT assay. To determine the effects ofdifferent concentrations of ketamine on the ratio of the apoptosis and cellcycle of the developping primary hippocampal neurons cultivated for12h with ketamine in vitro by flow cytometer and Hoechst33258immunofluorescencestaining.Methods: The immature hippocampal neurons cultivated for4-5d days invitro were undergone different concentrations of ketamine(0.1uM,1uM,100uM,300uM,1mM), and then cultivated for3h,6h,12h and24hrespectively. In order to correct the MTT value, blank groups were designed ineach experiment. The neurons were randomly grouped as control group anddifferent concentrations of ketamine groups. Then the MTT data of differentgroups were dected. Cell viability rate was derived from the formula asRate=(contorl group-blank group)/(experimental group-blank group).According to the results of MTT, the immature hippocampal neurons wereundergone different concentrations of ketamine as same as before andcultivated for12h further. According to formulary requirement,the samples ofthe all groups were prepared, and then the cell cycle and apoptosis rate of allgroups were analyzed by flow cytometer. In order to verify the results of FCM,apoptotic morphology of the all groups were detected by the Hoechst33258immunofluorescence staining.Results:1. The effect of ketamine on the hippocampal neurons viabilityhad time and concentration interaction (F=6.227, P<0.01). The difference ofthe effects of different ketamine's action time on the vitality of the neuronswas significant (F=6.750, P<0.01), in which300uM and1mM ketamine had asignificant difference (F=4.192, P<0.05and F=44.444, P<0.01), while nosignificant difference was observed among other concentrations due todifferent durations. On the other hand, the neural vitality had a significantdifference among different concentrations when the exposure duration was thesame (F=136.068, P<0.01), and in each same duration, compared with thecontrol group, effect of300uM and1mM ketamine on hippocampal neuronsvitality had a significant difference. The inhibition of neurons vitality was themost serious when neurons underwent1mM ketamine to culture for24h, andneurons vitality was down to60%. Interestingly,0.1and1uM ketamine at24hand12h had a positive effect of hippocampal neurons vitality respectively (P<0.05).2. The apoptosis rate had significantly different among each group(F=80.507, P<0.01). There were statistic differences in apoptosis rate of theneurons exposed to100,300and1000uM ketamine compared with those ofthe control group, and the apoptotic rate of cells was dose dependent.According to cell cycle, G₀/G₁, S and proliferation index of hippocampalneurons were significantly different among each group (F=109.192andP<0.01, F=57.342and P<0.01, F=108.726and P<0.01). There were statisticdifferences in G₀/G₁, S and proliferation index of10,100,300and1000uMketamine compared with those of the control group, and these differenceswere also dose dependent. However, there were no significant difference inG₂/M of hippocampal neurons among each group(F=2.463, P=0.077).3. Withthe increase of ketamine concentration, more and more neurons appearedtypical apoptotic morphology. Firstly, nucleus became irregular with a slightlyserrated border, and then the peripheral chromatins aggregated into densemasses, and finally nucleus broke into fragments known as apoptotic bodies.Conclusions: The inhibitions of Ketamine to the immature hippocampalneurons are concentration-and time-dependent. With the increase ofconcentration and time, the viabilities of neurons are significantly decreased tocompare with those of the control group. The viabilities of neurons undergothe greatest depression when the concentration of ketamine is1mM. Highconcentrations ketamine induces the increase of apoptosis in the immaturehippocampal neurons. Ketamine can promote neurons from G₀/G₁phase to Sphase but not to G₂/M phase. The proliferation of neurons is arrested in Sphase.Part â…¢ Effects of ketamine on spontaneous calcium oscillations ofdevelopping hippocampal neuronsObjective: To investigate the effects and mechanism of ketamine withdifferent concentrations on spontaneous calcium oscillations of developpingprimary hippocampal neurons in vitro.Methods: Primary cultivated hippocampal neurons on4th-5th werecleaned three times with Krebs-Ringer, and then these neurons were incubated for30min with diluent of Fluo-4AM at37oC, and then washed three timesagain to remove non-specific binding. The slide of hippocampal neuronsloaded with Fluo-4AM was laid in perfusion slot. Laser confocal microscopewas focused to make images of hippocampal neurons sharper. Argon ion laserwas used to excite the Fluo-4AM fluorescence of hippocampal neurons. Scanfrequency and mode was set as one per second and continuous scanningrespectively. Neurobasal culture medium and neurobasal culture mediumincluding the drugs studied were used to perfuse the hippocampal neurons oneafter another. There was one min drugs washout period between the twoperfusions.The time-lapse recordings of Ca²⁺signals in hippocampal neuronswere2min for each application of different chemicals. A semi-quantitativemeasurement was used to analysis spontaneous calcium oscillations ofdevelopping primary hippocampal neurons in vitro. Changes in thefluorescence of [Ca²⁺]i were represented by changes in the relative Fluo-4AMfluorescence (F/F₀), where F is the fluorescence at time t and F₀is the averageof the lowest fluorescence value in t-10and t+10s. The spontaneous calciumoscillation was deemed to occurr when F/F0exceeded1.2. The drugs studiedincluded ketamine,NMDA and MK801.Results:1. It is showed typical calcium oscillations in the neurons randomlyselected with a frequency of0.042±0.006Hz and amplitude of2.01±0.07.2.NMDA(100uM) could enhance amplitude but not frequency of calciumoscillations (P<0.01). MK801(40uM) could inhibit the frequency andamplitude of Ca²⁺oscillations (P<0.01).3. Compared with control group, therewas no difference in the effect of ketamine of1uM to10uM on the frequencyand amplitude of Ca²⁺oscillations. With the increase of ketamineconcentrations, the amplitude became more and more depression afterconcentration of ketamine reached30uM. On the other hand, with the increaseof ketamine concentrations, the frequency became more and more decreasewhen ketamine concentration reached300uM. When ketamine concentrationreached3000uM, spontaneous Ca²⁺oscillations were completely suppressed.4.With the increase of NMDA concentrations, it was showed more and more reversible enhancement in the frequency and amplitude of neurons undergone1000uM ketamine. They were completely reversed when NMDAconcentration reached1000uM.Conclusion:1. There is typical calcium oscillations in the developpinghippocampal neurons under laser confocal microscope.2. The calciumoscillations of immature hippocampal neurons could be inhibited by ketamineconcentration-dependent.3. The inhabition of calcium oscillations induced byketamine could be completely reversed by NMDA.Part â…³ Role of PKCγ-ERK signalling pathway in the neurotoxicity ofimmature hippocampal neurons induced by ketamineObjective: To investigate the effect of NMDA on apoptosis ofdevelopping hippocampal neurons induced by ketamine with FCM and todetect the role of PKCγ-ERK signalling pathway in the neurotoxicity ofketamine by western blot and immunocytochemistry.Methods: The neurons cultivated for4-5d days in vitro were divided intofour groups, the control group(C),300μM ketamine group(K),300μM ketamineplus100μM NMDA group(K+N) and100μM NMDA group(N). All groupswere cultured for12h, the early and late apoptosis rates in each group weredetected by the Annexin-V/Propidium iodide (PI) double-labeled flowcytometry. Immunohistochemistry and Western blot analysis were used todetect the expression levels of pPKCγ, tPKCγ, pERK1/2, tERK1/2and Bcl-2in each group.Results:1. Both early and late apoptosis rates in K group weresignificantly more increase than those in the control group (P<0.01, P<0.01).Compared with K group, both early and late apoptosis rates of K+N groupwere lower (P<0.01, P<0.01).However, both early and late apoptosis rates inK+N group were still higher than those of C group (P<0.01, P<0.05).2. Theexpressions of pPKCγ,tPKCγ, pERK1/2, tERK1/2and Bcl-2in K group weresignificantly lower than those of the control group.These protein expressionsof K+N group is higher than those of K group and still significantly lower thanthose of the control group.The data above were derived from the analyses of the immunohistochemistry assay.3. There was a similar changing tendency asthe results above in these protein relative expressions according to Westernblot analyses. The difference of the results was that the relative expressions oftERK₁/2and Bcl-2in K+N group are no longer lower than those of thecontrol group.Conclusions:1. Ketamine with300uM cultured12h could induce theincrease of apoptosis rate in immature hippocampal neurons.NMDA plays asignificant neuroprotective role in the neurotoxicity induced by ketamine.2.Ketamine could inhibit of PKCγ-ERK signalling pathway, which takes part inthe neurotoxicity mechanism of ketamine.Part â…¤ Effects of ketamine on hippocampus neuronal apoptosis in infantrats and the future learning and memorizing abilityObjective: To observe whether different dose ketamines that wererepeatedly administered to infant mice by intraperitoneal injections can triggerapoptosis in hippocampal neurons and whether the learning and memorizingability was impaired in later adulthood.Methods: Seventy-six infant rats were randomly assigned into fourgroups receiving intraperitoneal injections of normal saline (control);ketamine of25mg/kg(Group K₁);ketamine of50mg/kg(group K₂) andketamine of75mg/kg (group K₃) respectively. Rats in different groups werereceived different dose of ketamine or normal saline by intraperitonealinjections once a day for3days. Throughout the anesthesia, the rats were keptin a neonatal incubator to maintain body temperature and provided with lowflow oxygen to reduce potential stressors.All pups were separated from theirmothers about250minutes after administration of ketamine or normal saline.Twenty-four hours after last injection, the number of apoptotic cells in theCA1, CA2, CA3region and gyrus dentatus was detected by TUNEL assay(n=5). To determine whether ketamine induce persistent learning andmemorizing deficits, the learning and memorizing abilities were tested byMorris Water Maze at2months of age (n=14).Results:1. The number of apoptotic cell per mm2in group K₃at CA1and gyrus dentatus were more than those in control group (P <0.01and P <0.01).2. In five days training period, compared with the control group, rats in groupK₃took longer time and longer swiming distance to find the hidden platformon the fourth and fifth training day. There was no significant difference on thefirst three days of this five days training period. In the probe trial, the escapelatency of group K₂and K₃was longer than that of control group(P<0.05,P<0.01).The times of crossing platform and ratio of time spent in the targetquadrant were fewer in group K₃than in control group(P<0.05,P<0.01).Conclusions: The apoptosis of hippocampal neurons in SD infant rats couldbe induced by injections of ketamine three times, and the learning andmemorizing ability is impaired in later adulthood, which is dose-dependent.